CN104327159B - Mycobacterium tuberculosis specific C D8+T cell epitope peptide P45 and its application - Google Patents

Mycobacterium tuberculosis specific C D8+T cell epitope peptide P45 and its application Download PDF

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CN104327159B
CN104327159B CN201410596705.4A CN201410596705A CN104327159B CN 104327159 B CN104327159 B CN 104327159B CN 201410596705 A CN201410596705 A CN 201410596705A CN 104327159 B CN104327159 B CN 104327159B
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CN104327159A (en
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马骊
刘苏东
罗微
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Southern Medical University
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Southern Medical University
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Abstract

The invention discloses a kind of mycobacterium tuberculosis specific C D8+T cell epitope peptide and its application.The amino acid sequence of the epitope peptide is RLIPFAAPPK, and the epitope peptide is high with HLA A*1101 molecules affinity, can activate CD8+T cells, induce it to breed and secrete IFN γ.The determination of epitope peptide of the present invention, theoretical foundation and new solution are provided for the research and development of tuberculosis vaccine, diagnostic reagent and medicine, preventing and treating lungy is significant.

Description

Mycobacterium tuberculosis specific C D8+T cell epitope peptide P45 and its application
Technical field
The invention belongs to immunological technique field, more particularly to mycobacterium tuberculosis specific C D8+T cell epitope peptide and It is applied.
Background technology
Tuberculosis is that induction, global infection rate highest infectious disease, case fatality rate are only second to after being infected by mycobacterium tuberculosis AIDS.Only 2012, the new tuberculosis patient of sending out in the whole world was up to 8,600,000, dead 1,300,000.The new feature of tuberculosis epidemic situation is：It is resistance to Medicine tuberculosis case occurs and is in the gesture of sprawling；Tuberculosis grows in intensity with AIDS coinfection situation.For this two classes patient, at present Have no effectively preventing measure.China is that tuberculosis bears one of most heavy country in the world, researches and develops the knot for being adapted to Chinese population Time and tide wait for no man for core disease vaccine and medicine.
Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is intracellular bacterial parasite, to its effective control Cellular immunity is depended on, especially T cell is immunized.T cell is broadly divided into CD4 in human body+T (Th) cells and CD8+T (CTL) two big subgroup of cell, numerous studies are it has been proved that both T cell subgroups all have weight for control tubercle bacillus affection Act on, wherein CD8+CTL is even more the main force for removing tulase.
The complete tulase of T cell None- identified and proteantigen, but identify and absorb and add by antigen presenting cell The tuberculosis epitope peptide discharged after work.Epitope peptide is the core part for causing immune response in antigen, also referred to as antigenic determinant, Its HLA molecule with antigen presenting cell surface combines to form peptide-HLA compounds and is delivered and expresses in antigen presenting cell Surface, and then activating T cell, induce t cell immune response.CD4+T cell identifies the epitope peptide of HLA II quasi-molecule submissions, and CD8+T cell identifies the epitope peptide of HLAI quasi-molecule submissions.Identification and its activation of the T cell to epitope peptide require epitope peptide with resisting Former presenting cell surface HLA molecules stable bond, and the affinity of peptide and HLA molecules is to form the key of peptide-HLA compounds, Therefore prepare and induce T cell immune medicine and vaccine, first step is the peptide for filtering out high-affinity, and then is identified With immunocompetent epitope peptide.
Epitope peptide can be completely short by artificial synthesized acquisition, R＆D cycle；Due to eliminating pathogen beyond epitope peptide Other harmful components, security are good；Multiple epitope peptides can also be used in series, booster immunization effect and reduction are immunized and escaped Ease.The tulase CTL epitope peptides being currently known mainly identify that mostly HLA-A*0201 is restricted, and right in American-European crowd In the Africa of TB endemic most serious and Southeast Asia crowd, unsuitable epitope peptide can be used.In China, HLA-A* 1101 be gene frequency highest HLA molecules in crowd, during identification HLA-A*1101 restricted CTL epitopes are adapted to for research and development The epitope peptide vaccine of state crowd has very important significance.
The content of the invention
It is an object of the invention to provide mycobacterium tuberculosis specific C D8+T cell epitope peptide.
Another object of the present invention is to provide mycobacterium tuberculosis specific C D8+T cell epitope peptide is preparing tuberculosis Application in disease vaccine, diagnostic reagent and medicine.
The technical solution used in the present invention is：
The invention provides high with HLA-A*1101 molecules affinity, CD8 can be activated+T cell, it is induced to breed and secrete The mycobacterium tuberculosis specific C D8 of IFN-γ+T cell epitope peptide, the amino acid sequence of the epitope peptide are：RLIPFAAPPK (SEQ ID NO.45) (i.e.：Arg-Leu-Ile-Pro-Phe-Ala-Ala-Pro-Pro-Lys).
The present invention combines Bioinformatics Prediction method and the displacement experiment of uv induction peptide, utilizes mycobacterium tuberculosis itself Antigen selection go out the epitope peptide with anti-tubercular, efficiently and accurately detect epitope peptide RLIPFAAPPK (SEQ ID NO.45) the affinity between HLA-A*1101 molecules.This is the restricted tuberculosis of HLA-A*1101 that is new, not being reported Mycobacterial epitope peptide, by tuberculosis patient CD8+T cell identifies extensively.Due to HLA-A*1101 genotype be in Chinese it is most wide The genotype of general carrying, the special HLA-A*1101 restricted CTL epitope peptides of mycobacterium tuberculosis, the present invention are not found at present The determination of epitope peptide, theoretical foundation and new solution party are provided for the research and development of tuberculosis vaccine, diagnostic reagent and medicine Case, preventing and treating lungy is significant.
Brief description of the drawings
Fig. 1 is candidate's epi-position peptide and HLA-A*1101 molecule affinity schematic diagrames.
Fig. 2 is the schematic diagram of the epitope inducing peptide tubercular T cell activation of 22 high-affinities and secretion of gamma-IFN.
Fig. 3 is that ELISPOT detects the schematic diagram that P45 induces tubercular's T cell secretion of gamma-IFN.
Fig. 4 is P45 mass spectral analysis figure.
Fig. 5 is that the lymphocyte proliferation assay of CFSE marks detects P45 induction tuberculars CD8+The result of T cell propagation Schematic diagram.
Embodiment
Primary structure of the invention according to antigen, using Immunoinformatics means, with CBS databases NetMHCcons Antigen Epitope Predictions software is carried out to the HLA-A*1101 restricted CTL epitopes peptide of antigen of mycobacterium tuberculosis albumen Forecast analysis, then using the uv induction peptide affinity that is combined with HLA-A*1101 molecules of displacement experimental check candidate peptide and Stability, screening obtain epitope peptide, identified by experiment in vitro, CTL epitope peptides P45 can induce CD8+T cell produces strong Cellullar immunologic response, secreting high levels IFN-γ, and induce CD8+Significantly propagation occurs for T cell.
Present invention is expanded on further with reference to specific embodiment.
Using bioinformatics method, HLA-A*1101 restricted epitope peptides are predicted：
According to the literature, choosing 94 has more strongly immunogenic antigen of mycobacterium tuberculosis albumen (as shown in table 1), The amino acid sequence of each antigen protein is obtained from GeneBank.
The candidate's antigen of mycobacterium tuberculosis albumen of table 1
MTB antigens
Rv0079 Rv1478 Rv2030c Rv3044
Rv0288 Rv1569 Rv2031c Rv3127
Rv0315 Rv1626 Rv2032 Rv3130c
Rv0350 Rv1733c Rv2034 Rv3131
Rv0440 Rv1738 Rv2108 Rv3132c
Rv0467 Rv1787 Rv2220 Rv3347c
Rv0475 Rv1788 Rv2244 Rv3353c
Rv0496 Rv1789 Rv2324 Rv3418c
Rv0577 Rv1790 Rv2380c Rv3420c
Rv0685 Rv1791 Rv2389c Rv3478
Rv0733 Rv1793 Rv2450c Rv3615c
Rv0754 Rv1813c Rv2468c Rv3619c
Rv0824c Rv1837c Rv2608 Rv3716c
Rv0831 Rv1860 Rv2620c Rv3803c
Rv0867c Rv1884c Rv2623 Rv3804c
Rv0932c Rv1886c Rv2626c Rv3873
Rv0934 Rv1908c Rv2627c Rv3874
Rv0978c Rv1926c Rv2628 Rv3875
Rv1009 Rv1956 Rv2629 Rv3914
Rv1130 RV1966 Rv2744c
Rv1169c Rv1980c Rv2770c
Rv1174c Rv1996 Rv2780
Rv1349 Rv2005c Rv2875
Rv1363c Rv2006 Rv2903c
Rv1411 Rv2029c Rv3020c
China is obtained from HLA gene frequency databases AFND (Allele Frequency Net Database, AFND) The frequency distribution information of each genotype in crowd HLA-A sites, wherein HLA-A*1101 gene frequencies are about 28%, are HLA-A positions Frequency highest individual gene type on point.
Use the HLA-A*1101 restricted CTL epitopes contained by bioinformatics prediction antigen protein, the calculation of use Method is the NetMHCcons in CBS databases.Prediction result is ranked up according to IC50 values, IC50 values are lower, illustrate epitope Peptide and HLA molecule affinity are higher.Thus, HLA-A*1101 restricted epitope peptides in 94 antigen proteins are predicted, and are sieved Wherein 50 epitope peptides of affinity highest are selected, as candidate, as shown in table 2.
The restricted candidate's epi-position peptides of 2 50 HLA-A*1101 of table
Candidate's epi-position peptide is numbered Amino acid sequence Antigen protein IC50(nM)
p1 MTNTLHSMLK(SEQIDNO.1) Rv3478 4.67
p2 KTVTFMPK(SEQIDNO.2) Rv2220 5.29
p3 TTLDAGFLK(SEQIDNO.3) Rv3130c 5.9
p4 VVMTTTLSK(SEQIDNO.4) Rv1569 6.16
p5 ISSGVFLLK(SEQIDNO.5) Rv2029c 6.4
p6 VSIPTLILFK(SEQIDNO.6) Rv3914 6.97
p7 SSMTRIAK(SEQIDNO.7) Rv1884c 7.16
p8 MLFSMHGELYK(SEQIDNO.8) Rv1196c 7.36
p9 TAMRVTTMK(SEQIDNO.9) Rv1009 7.69
p10 STIDEFAK(SEQIDNO.10) Rv0496 7.73
p11 TTSPIPLK(SEQIDNO.11) Rv3347c 7.94
p12 AVMAGIVRAAK(SEQIDNO.12) Rv3130c 8.07
p13 VSIARALLK(SEQIDNO.13) Rv1349 8.11
p14 AVAAPAFAEK(SEQIDNO.14) Rv1790 8.16
p15 GTHPTTTYK(SEQIDNO.15) Rv1980c 8.25
p16 TTSNVSVAK(SEQIDNO.16) Rv0867c 8.25
p17 AVNTLFEK(SEQIDNO.17) Rv3873 8.34
p18 KVNRFPDPK(SEQIDNO.18) Rv3131 8.34
p19 MTSGSSSGFK(SEQIDNO.19) Rv3347c 8.34
p20 GTQAVVLK(SEQIDNO.20) Rv1980c 8.52
p21 SVNNYQASK(SEQIDNO.21) Rv2006 8.61
p22 ATIAKFQK(SEQIDNO.22) Rv0467 8.8
p23 MTSLDFNK(SEQIDNO.23) Rv1363c 8.8
p24 VVMPVLKK(SEQIDNO.24) Rv0824 8.94
p25 GTFKSVAVK(SEQIDNO.25) Rv0440 9.65
p26 AVARLVAISK(SEQIDNO.26) Rv3130c 9.86
p27 TTLTAAITK(SEQIDNO.27) Rv0685 9.86
p28 RVLGANYK(SEQIDNO.28) Rv1908c 10.07
p29 ATFAEIGHK(SEQIDNO.29) Rv2324 10.69
p30 KTFGFGFGR(SEQIDNO.30) Rv1908c 11.11
p31 KTQGPGAWPK(SEQIDNO.31) Rv2389c 11.6
p32 KTYCEELK(SEQIDNO.32) Rv1980c 11.66
p33 SVQMTLSK(SEQIDNO.33) Rv1411 11.79
p34 TVFDYHNENAK(SEQIDNO.34) Rv2108 11.79
p35 VSNAVRHAK(SEQIDNO.35) Rv3132c 12.24
p36 CVANMPASVPK(SEQIDNO.36) Rv2780 12.58
p37 RVQTTVLK(SEQIDNO.37) Rv2108 12.64
p38 VSNSLVAHMK(SEQIDNO.38) Rv2780 12.64
p39 AVAVVLHK(SEQIDNO.39) Rv2380c 12.99
p40 HTVALFLDK(SEQIDNO.40) Rv2032 12.99
p41 AIAADPSFK(SEQIDNO.41) Rv1956 13.06
p42 MTQWLIEEK(SEQIDNO.42) Rv2030c 13.13
p43 GTSDNFQK(SEQIDNO.43) Rv0932c 13.2
p44 KAFAPAHAGK(SEQIDNO.44) Rv1130 13.71
p45 RLIPFAAPPK(SEQIDNO.45) Rv1787 13.71
p46 TVINASRFK(SEQIDNO.46) Rv2380c 13.79
p47 VVNKIRGTFK(SEQIDNO.47) Rv0440 13.94
p48 ATTAFGAK(SEQIDNO.48) Rv1966 14.09
p49 QANAHGQK(SEQIDNO.49) Rv1037c 326.55
p50 NNMAQTDSAV(SEQIDNO.50) Rv1037c 29745
Experimental method is replaced using uv induction peptide, detects the affinity between candidate's epi-position peptide and HLA-A*1101 molecules And combination stability：
Experimentation is as follows：
Under ultraviolet irradiation, photosensitivity epitope peptide is broken the peptide-HLA compounds of the epitope peptide containing photosensitivity, Depart from from HLA molecules, at this moment toward candidate's epi-position peptide to be detected is added in reaction system, if candidate's epi-position peptide and HLA points Sub- affinity is high, then can form the epitope peptide-HLA compounds of new stabilization.Using anti-beta 2m antibody capture HLA molecules, Epitope peptide-HLA the complex concentrations that ELISA detections are newly formed, evaluate the affinity size between epitope peptide and HLA molecules.Altogether The epitope peptide (being more than 100%) 22 that there is high-affinity with HLA-A*1101 is selected, as shown in Fig. 1 and table 3.
Table 3 and HLA-A*1101 has 22 epitope peptides of high-affinity
ELISpot experiment 22 epitope peptide inducing T cell secretion of gamma-IFN of detection, the immune effect of preliminary identification epitope peptide Should：
Experimentation is as follows：
Using density gradient method separation HLA-A*1101 genotype tubercular's PMNCs (peripheral blood mononuclear,PBMC).The 20ml peripheral bloods of collection are diluted one times with PBS, are slowly added to separation of lymphocytes Above liquid, both volume ratios are 2:1,30min is then centrifuged with 800g/min rotating speeds, liquid is divided into three layers after centrifugation, wherein PBMC, into tunica albuginea shape, PBMC is carefully drawn with dropper into new centrifuge tube between first layer and the second layer, add 5 times with Upper volume PBS is washed, and 800g/min centrifugation 10min, repeated washing once, is frozen carefully with frozen stock solution (90%FBS+10%DMSO) Born of the same parents, liquid nitrogen storage.
ELISpot is tested：Recovery HLA-A*1101 genotype patient PBMC, at 37 DEG C, 5%CO2Stood overnight in incubator. Spend the dead cell in dead cell magnetic bead removal PBMC.Cell is resuspended to 1.25 × 10 in cell count, complete medium6Individual/mL, 200 μ L/ holes cell suspensions are spread into ELISpot plates (i.e. 2.5 × 105Individual/hole), totally 48 hole.By 22 candidate peptides shown in table 3 It is separately added into by 10 μ g/mL concentration in 44 holes, a kind of peptide is added per hole, every kind of peptide spreads 2 holes, and 2 Positive control wells are separately added into 4 μ G/mLPHA, 2 negative control holes are not added with any stimulation, fill full training.After each hole has added sample respectively, culture plate is placed in cell In incubator, 37 DEG C, 5%CO2Cultivate 24h.ELISpot plates are taken out, PBS is washed 5 times, and the anti-IFN-γ of enzyme mark is added after patting dry and is resisted Body 100ul, 2h is incubated, PBS is washed 5 times, and developer 100ul is added after patting dry, and after obvious spot occur in Positive control wells, is used Ultra-pure water terminating reaction；Dry, read plate, count, correction.Peptide-specific T-cell frequency is with SFC/106Represent.
Positive reaction criterion is：①SFC/106>50,2. peptide stimulation hole spot number is more than or equal to negative control hole Twice.Meet 1. that 2., reaction and judgement is the positive simultaneously.
Identification reactions of 23 HLA-A*1101 type tubercular PBMC to epitope peptide is counted, i.e. patient P BMC is to epitope peptide Identification frequency.The detection Ren Shuo ╳ 100% of peptide discrimination=positive reaction number/total.Filtering out at least can be by 20% patient The epitope peptide of identification, as a result as shown in Figure 2.Fig. 3 is that ELISPOT detects epitope peptide P45 induction tuberculosis patient T cell secretions IFN- γ result schematic diagram.
The lymphocyte proliferation assay detection epitope peptide P45 inductions CD8 of CFSE marks+T cell is bred：
Experimentation is as follows：
Recovery HLA-A*1101 type tubercular PBMC, stand overnight.Dead cell, meter are removed using dead cell magnetic bead is removed Number, cell is resuspended to 1 ╳ 10 with PBS after centrifugation7Individual/mL, 5 μM of CFSE marks are added, lucifuge is incubated 10min after mixing.Add 5 The complete medium (10%FBS+RPMI-1640) of times volume precooling terminates dye marker.300g centrifuges 10min, with complete training Base washing is supported, is repeated twice.Count again, cell is resuspended to 1 ╳ 10 with complete medium6Individual/mL.Cell suspension is spread into 96 In the U floor cells culture plates of hole, 200 μ L/ holes, totally 8 hole, each hole of experimental group are separately added into 10 μ g/mL epitope peptides P45 (Fig. 4 P45 Mass spectral analysis figure), Positive control wells add 4 μ g/mLPHA, and negative control hole is not added with any stimulant, supplies full training.Glimmering Viewed under light microscopy, determine that cell is marked (green fluorescence) by CFSE.37 DEG C, 5%CO248h is cultivated in incubator.Each Kong Zhongfen 100IUIL-2 is not added, continues culture to 7d, takes out cell, anti-CD8 antibody, flow cytometry inspection are marked after washing Survey.
According to the characteristics of CFSE dye markers, dyestuff is equably assigned to daughter cell with cell propagation from parent cell In, fluorescence intensity halves.So the cell of weak CFSE fluorescence, the cell after as breeding.Epitope inducing peptide CD8+T cell increases Grow CD8 of the ability by weak CFSE fluorescence+T cell/CD8+T cell represents.Testing result is as shown in Figure 5.
Epitope peptide P45 by above scheme Screening and Identification, specifically it can be identified by HLA-A*1101 types tubercular, And induce CD8+T cell is bred and secretion of gamma-IFN, and reason is provided for the research and development of tuberculosis vaccine, diagnostic reagent and medicine By basic and new solution, preventing and treating lungy is significant.
<110>Nanfang Medical Univ
<120>Mycobacterium tuberculosis specific C D8+T cell epitope peptides P45 and its application
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Claims (3)

1. mycobacterium tuberculosis specific C D8+T cell epitope peptide P45, the amino acid sequence of the epitope peptide are：RLIPFAAPPK (SEQ ID NO.45)。
2. the mycobacterium tuberculosis specific C D8 described in claim 1+T cell epitope peptide P45 is preparing diagnostic reagent of tuberculosis And the application in treatment tubercular drugs.
3. the mycobacterium tuberculosis specific C D8 described in claim 1+T cell epitope peptide P45 is preparing tuberculosis prophylaxis epidemic disease Application in seedling.
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